The development of the diagnostic field in the last few decades had a meaningful influence on the improvement occurring in health and medical fields. The progress in diagnostics brought forth a wide variety of efficient and reliable diagnostic tools, which are still considered expensive, complicated to use even for the trained personnel, and available mainly in large medical centres. As such, there is a continuous need worldwide for developing new and improved diagnostic and monitoring tools and techniques, which will at the same time be portable, field operational and provide fast, accurate, direct, quantitative, low cost tests for both viral and bacterial infections, and tumour diagnostics. Over the years the progress in technological devices has led to the development of several prototype biosensors enabling simple, sensitive, specific, and safe detection systems. However, a great potential still lies in the development and improvement of the bioassays and biosensors niche with new and better methods and devices.
Bioassays and biosensors are based on a specific recognition of a particular analyte with any recognition element, such as antibody, antigen, DNA, enzyme, bioreceptor or aptamer, which combines with its specific DNA substrate, antigen or hapten to give an exclusive complex. Antigens are generally high molecular weight proteins, polysaccharides, lipids and polypeptides, which can be detected in different immunoassay configurations. Smaller organic molecules (haptens), such as drugs, simple sugars, amino acids, small peptides, phospholipids, or triglycerides can also be detected, provided that they are chemically coupled to a carrier protein or other synthetic matrices. Thus, just about any analyte can be spotted by the immune system triggering the specific antibody production. Nonetheless, the small molecules (haptens) do not need to be conjugated to a carrier protein or other synthetic matrices, if aptamers are used for detection, instead of antibodies in ‘non-sandwich’ type assays.
Many types of bioassays have been recently applied to clinical diagnostics, environmental analysis and food safety assessment. Most of them involve synthetic conjugates comprising radioactive, enzymatic, fluorescent, chemiluminescent or visually observable metal sol tags, and specially designed reactor chambers, such as microplates. These assays although being quantitative, such as ELISA, follow long protocols and may takes hours and many reaction steps to complete. They suffer from the relatively high cost and cumbersome procedures, which require expensive instruments and trained personnel.
On the other hand, the lateral flow immunoassay, which is also known as a “strip” test, is an example of a widespread test that is simple to perform by almost anyone and produces results more rapidly than any traditional laboratory-based testing. The coloured lines across the strip can take as little as a few minutes to develop. This area of diagnostics has grown dramatically in recent years, with the most common and well-known of these being the home pregnancy test. Lateral flow immunoassay holds a great diagnostic advantage in the fact that it is cheap, simple for operation, rapid (just a few minutes) and portable. It typically requires little or no sample or reagent preparation. The strips are very stable and robust, have a long shelf life and do not usually require refrigeration. This technique has a wide range of medical diagnostic applications, e.g. ascertain pregnancy, failure of internal organs (e.g. heart attack, renal failure or diabetes), infection or contamination from specific pathogens. In some cases, diseases, such as cancer, can be rapidly detected using the strip test by analysing the blood stream for tumour specific markers, typically, specific antibodies.
The principle of a lateral flow immunoassay relies on the competition for binding sites on a polymer or metal particles. Antibodies that are raised to a specific target are bound to metal nanoparticles or dyed polymer particles. These particles are then applied using an immersion procedure onto a release pad in order to produce a stable particle reservoir for release onto a nitrocellulose-based membrane. Two lines of reagents are immobilised onto the membrane. The target reference or test line comprises a conjugate that can specifically bind the target to be identified and the other, the control, is a line of anti-anti-target antibodies. The release pad and membrane are assembled together with an absorbent pad. The sample is initially added to the adsorbent pad and then the strip is left for a few minutes with the result read directly by eye, looking for the presence of coloured lines. These kits are relatively cheap to make. They also have a long shelf-life and are fully disposable. This technology is, therefore, ideally suited to any rapid diagnostics.
Lateral Flow Immunoassay
R. Wilson (2008) in “The use of gold nanoparticles in diagnostics and detection”, Chemical Society Reviews 37(9): 2028-2045, reviewed that in their most common form, lateral flow immunoassays consist of porous white membranes striped with a line of antibodies or antigens, and interfaced with antibodies conjugated to labels that can be seen with the naked eye. Reference is initially made to FIG. 1 showing a prior art strip. It should be noted that FIG. 1 relates to prior art knowledge, and as such it merely constitutes a reference for better understanding of the present invention.
Design of the lateral flow device (strip), according to FIG. 1, shows the four major components of the strip (sample pad, conjugate pad, nitrocellulose membrane and wick). TL and CL stand for the test and control lines, respectively. The liquid sample containing the target analyte molecule or entity is applied to the nearest end of the strip, the sample pad or absorbing pad which is located on top of the conjugate pad. The latter contain colorimetric metallic nanoparticles, such as gold nanoparticles (GNPs) or polymeric (e.g., latex) microspheres, coated with the antibodies, which are specific to the target analyte molecules (antigens). The sample migrates through the nitrocellulose membrane by capillary action.
The hydrophobic nitrocellulose membrane serves as a bedding (solid support), onto which anti-target analyte antibodies are immobilised in bands across the membrane in specific areas of the membrane where they capture the analyte and the conjugate as the latter migrate. Essential in the lateral flow immunoassay is the migration of a liquid sample, or its extract containing the analyte of interest, through various zones of the strip where binding molecules have been immobilised that exert more or less specific interactions with the analyte.
As shown in FIG. 1, at least two lines are laid down on the strip: a test line (TL) and a control line (CL), which have both been pre-treated with specific antibodies or antigens, and which is the standard for the commercially available lateral flow strips. The control line may contain either antigens or antibodies that are specific for the conjugate antibodies. Generally, the TL binds the analyte while the CL binds the capture antibody or antigen attached to the red-coloured, coated gold nanoparticles. These lines are usually closer to the wicking pad than to the conjugate pad in order to improve the overall performance of the lateral flow immunoassay. Some lateral flow assays may have more than one test line, but each additional test line greatly increases the complexity of the immunosensor, and thus increases cost.
After absorbing the liquid sample onto the sample pad, the liquid moves into the conjugate pad by capillary action, rehydrates the labelled conjugate particles and allows the mixing of these particles with the absorbed liquid sample. The labelled conjugate interacts with the specific analyte contained in the sample, thereby initiating the intermolecular interactions, which are dependent on the affinity and avidity of the reagents. Then it starts migrating towards the test line capturing and recognising the binding analyte, where it becomes immobilised and produces a distinct signal for example, in the form of a coloured line, indicating the test is positive. Excess reagents move past the capture lines and are entrapped in the wick pad, which is designed to draw the sample across the membrane by capillary action and thereby maintain a lateral flow along the chromatography strip.
Notwithstanding the immediate success of the lateral flow strips, their current applications leave much to be desired. A distinct signal at control line may indicate a proper flow of the body liquid through the strip. Depending upon the analytes present in the sample and on the type of the immunoassay performed, the coloured reagent becomes bound both at the test line and at the control line, or, alternatively, only at the test line. Thus, the results are interpreted on the reaction matrix as the presence or absence of lines of captured conjugate, and are read either by eye or using a reader. These results are unfortunately binary (“yes or no”) and do not provide any quantitative measure of the analyte present in the sample. In other words, the major disadvantage of the lateral flow devices is that they are only capable of producing qualitative or semi-quantitative measurements, while a quantitative option can be obtained only using a special and additional instrumentation such as a colorimeter.
In addition, the lateral flow strips suffer from a large number of false positive results. Some lateral flow immunoassays are competitive assays, which differ from the antibody sandwich immunoassays in that the conjugate pad contains antibodies that are already bound to the target analyte. If the target analyte is present in the sample, it will not be able to create the complex with the conjugate and hence, will remain unlabelled. Competitive immunoassays are most suitable for testing small molecules, such as toxins and hormones, and unable to bind to more than one antibody simultaneously. For example, if an excess of the unlabelled analyte is not present, a slightly coloured line may appear in the test line, indicating an inconclusive result.
Failure of the lateral flow immunoassay to provide quantitative results has prompted an urgent need for a new bioassay and biosensor that would allow quantitative measurements within the same simple and miniaturised framework of the current lateral flow strip while maintaining a rapid diagnostics, long shelf life and an easy handling by untrained personnel. With the market value of lateral flow immunoassay kits and devices estimated at approximately 2.1 billion dollars, and with over 200 companies worldwide producing a range of testing formats, many attempts have been made to improve upon these devices. Thus, there is a long-time need for development of a new format for lateral flow assay, which would be quantitative, more sensitive and show no false positive results.
It is therefore an object of the present invention to solve the problems associated with the modern lateral flow assays and biosensors, and to perform the quantitative measurements within the same platform. The present invention solves these problems by successful combination of the lateral flow technology with the electrochemical method in one assay and in a single device. Such integration of the electrochemical method into the lateral flow technology, as described below, produces a quantitative, quick and portable novel platform for a biosensor.
I. Ojeda et al (2012), in “Electrochemical immunosensor for rapid and sensitive determination of estradiol”, Analytica Chimica Acta 743: 117-124, describes the preparation of an electrochemical immunosensor for estradiol based on the surface modification of a screen printed carbon electrode with grafted p-aminobenzoic acid followed by covalent binding of streptavidin and immobilization of biotinylated anti-estradiol (anti-estradiol-biotin).
U.S. Pat. No. 6,478,938 provides an electrochemical membrane strip biosensor, which combines the immunochromatographic method and electric conductivity detection technology. This biosensor uses gold nanoparticles for the measurement of metal conductivity. The metal colloids generate the quantitative signal in the electrochemical signal generating membrane pad. Thus, the analytical signal of the biosensor is based on metal conductivity of gold nanoparticles.
U.S. Pat. No. 7,300,802 relates to a biosensor comprising a regular membrane strip chromatographic assay system with four membrane pads as described above, and an additional membrane pad for the supply of substrate solution for enzyme. In addition, the strip has a cross-arrangement of two groups of the membrane pads and hence, includes a pad for absorption of vertical flow medium and a pad for absorption of horizontal flow medium. The biosensor shows successive cross-flow procedure for immune reaction and enzymatic reaction, and it uses HRP enzyme to provide the analytical signal. It is a complicated multiple-step test that includes the immunological detection together with the enzymatic reaction, resulting in the electrochemical signal measured with a screen printed electrode.
US 2004/0106190 discloses a flow-through assay device for detecting the presence or quantity of an analyte residing in a test sample. This device contains a fluidic medium, which is in communication with an electrochemical affinity biosensor. The latter utilizes detection and calibration working electrodes that are capable of generating a measurable detection current and communicate with affinity reagents, such as redox mediators and capture ligands. The amount of the analyte within the test sample is determined by calibration of the detection current with the calibration current. This sensor uses a redox enzyme to provide the electrochemical signal.
Dan Du et al (2012), in “Integrated Lateral Flow Test Strip with Electrochemical Sensor for Quantification of Phosphorylated cholinesterase: Biomarker of Exposure to Organophosphorus Agents”, Analytical Chem. 84: 1380-1385, describes an integrated lateral flow immunoassay strip with an electrochemical sensor device for quantification of exposure to pesticides (organophosphates) and nerve agents. This biosensor is based on the use of antibody to selectively capture the enzyme for enzyme activity assay. The test strip coupled with a portable electrochemical analyser is used for immunoreaction and selective separation of the enzyme from biological samples. The biosensor provides the measurement of the total amount of enzyme (including inhibited and active).
The novel biosensor of the present invention (ELFB—electrochemical lateral flow biosensor) has a totally new design and can easily be applied to various biological systems for the detection of bacterial, parasitic and viral infections, tumours, as well as toxins explosives and other pollutants in wastewater and in biological liquids. All the aforementioned prior art biosensor devices significantly differ from the ELFB in the following:                1. The prior art biosensors do not perform the detection in a single step. The main advantage of the ELFB is the single-step analyte detection, since there is no requirement to chemically treat or label the sample prior to the measurement. On the other hand, the aforementioned assays, which use the redox enzymes, will always require an additional step, in which the substrate will be added.        2. Their electrochemical sensor uses redox enzymes for evaluation of the analyte concentration. The ELFB does not use enzymes. In the ELFB, the amperometric signal is produced by reduction of an electrochemically active component (EAC), the role of which in the electrochemical system is to transfer electrons to the electrode corresponding to its redox potential.        3. The overall design of the prior art biosensors places the detection area in the middle of the nitrocellulose membrane. Because the detection of the analyte is performed during the capillarity flow along the nitrocellulose membrane, the longer the membrane, the better will be the separation and detection, which results in less false positive readings. Hence, the ELFB is designed in a way that it will allow the longest flow time by placing the detection area (modified screen printed electrode) at the end of the strip.        4. The prior art electrochemical biosensors do not use the electroactive beads or Particles. The ELFB novelty is also based on use of the electroactive beads or particles. These beads or particles can be either pre-coated with the electrochemically active component (EAC) or alternatively, can be made electroactive by themselves.        